Yan, Y. et al. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23, 781-790

Department of Anatomy, University of Wisconsin-Madison, 1500 Highland Avenue, Madison, Wisconsin 53705, USA.
Stem Cells (Impact Factor: 7.13). 05/2005; 23(6):781-90. DOI: 10.1634/stemcells.2004-0365
Source: PubMed

ABSTRACT How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Treatment of hESC-derived Sox1+ neuroepithelial cells with FGF8 and SHH resulted in production of tyrosine hydroxylase (TH)-positive neurons that were mostly bipolar cells, coexpression with gamma-aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8 treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of TH+ neurons characteristic of midbrain projection DA neurons with large cell bodies and complex processes and coexpression of En1. This suggests that one mechanism of generating neuronal subtypes is temporal availability of morphogens to a specific group of precursors. The in vitro-generated DA neurons were electrophysiologically active and released DA in an activity-dependent manner. They may thus provide a renewable source of functional human DA neurons for drug screening and development of sustainable therapeutics for disorders affecting the DA system.

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    • "The pluripotency was tested by teratoma assay (Hu et al., 2010). For dopamine neuron generation, primitive neuroepithelia at day 10 of iPSC differentiation were treated with SHH (C-24/25, R&D, 200 ng/ml) and FGF8 (R&D, 100 ng/ml) from days 14–28 (Yan et al., 2005). The neural progenitors were then cultured on a laminin substrate in low SHH (50 ng/ml) and FGF8 (50 ng/ml) until day 42 for immunocytochemical analysis and transplantation (Yang et al., 2008). "
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    ABSTRACT: The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies.
    Cell Reports 03/2013; 3(3). DOI:10.1016/j.celrep.2013.02.016 · 7.21 Impact Factor
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    • "Abbreviations: bFGF, basic fibroblast growth factor; BMP, bone morphogenic protein; DAPI, 4 0 ,6 0 -diamidino-2-phenylindole dihydrochloride; EB, embryoid body; ECM, extracellular matrix; FBS, fetal bovine serum; FGF8, fibroblast growth factor 8; HepG2, hepatocellular carcinoma cell line; hESCs, human embryonic stem cells; MEF, mouse embryonic fibroblast; Nurr1, nuclear receptor related 1 protein; Oct4, octamer-binding transcription factor 4; SHH, sonic hedgehog; Sox2, SRY (sex determining region Y)-box 2; SSEA4, stage-specific embryonic antigen 4; TH, tyrosine hydroxylase in the hESC lines used. Experimental protocols for differentiation of hESCs to dopaminergic cells range from using animal origin nutrient supplements and induction strategies to chemical inducers, undefined media components , hormones and developmental cues in multistep processes (Schulz et al., 2004; Zeng et al., 2004; Yan et al., 2005; Iacovitti et al., 2007; Sonntag et al., 2007; Shimada et al., 2009; Zhang and Zhang, 2010). Use of conditioned media or co-culture system with mouse stromal cell lines PA6, MS5 and liver hepatocellular carcinoma cell line HepG2 in differentiation protocol are liable to show inconsistent results on account of passage-linked differences. "
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    ABSTRACT: Although there are several reports on differentiation of human embryonic stem cells to dopaminergic neurons, notable heterogeneity exists in the reported yields of tyrosine hydroxylase (TH)-positive cells. For benchmarking performance and efficiency standards in future applications of hESC-derived dopaminergic neurons, there is thus a dire need of well-defined directed differentiation protocols. Pal et al. [Pal et al. 2009 Exp Biol Med (Maywood) 234:1230-3] demonstrated predisposition of HUES9 towards ectodermal lineage, but the directed differentiation of HUES9 to dopaminergic neurons has not yet been reported. Therefore, we report here a simple two-step protocol using suitable ECM and serum-free induction medium for generating dopaminergic cells from HUES9-derived embryoid bodies. Flow cytometry analysis of the neural progenitors obtained after the first step gave an enriched yield of cells immune-positive for nestin (99.6 ± 0.1%), musashi12 (98.1 ± 1.5%) and Sox2 (95.4 ± 2.6%). Most of these cells also expressed the proliferation marker Ki67 (83.8 ± 1.5%), whereas the presence of the undifferentiated stem cell marker Oct4 was negligible. In the second step, when these neural progenitors were exposed to midbrain cues sonic hedgehog and fibroblast growth factor 8 along with bFGF, the differentiated cells showed an upregulation of dopaminergic-related transcription factors Nurr1 and Engrailed1. Immunocytochemistry and flow cytometry analysis showed that these differentiated cells were positive for the mature neuronal marker Map2ab (96.2 ± 1.5%) and dopaminergic neuronal marker TH (71.9 ± 4.4%). Thus, the data demonstrate novel findings of the directed differentiation of HUES9 to dopaminergic neurons using well-defined serum-free nutrient supplements.
    Cell Biology International 01/2013; 37(1):54-64. DOI:10.1002/cbin.10012 · 1.64 Impact Factor
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    • "This approach uses a combination of bone morphogenetic protein 4 inhibitors (such as Noggin or Dorsomorphin) and inhibitors of Lefty/activin/TGFβ pathway (such as SB431542) to improve the efficiency of the differentiation (Chambers et al., 2009). At present, differentiation protocols do not exist for the generation of all cell types of the central nervous system (CNS), however over the past decade progress has been made for directed differentiation of hESCs into several neural cell types of the CNS (Suter and Krause, 2008; Liu and Zhang, 2011; see also in the same issue Martinez et al., 2012) including specific subtypes of neurons (Wichterle et al., 2002; Ying et al., 2003; Yan et al., 2005; Lee et al., 2010), oligodendrocytes (Hu and Zhang, 2009, 2010; Hu et al., 2009), astrocytes (Krencik et al., 2011; Liu and Zhang, 2011), and retinal cells (Meyer et al., 2009, 2011; Osakada et al., 2009; Lamba and Reh, 2011). "
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    ABSTRACT: The ability to generate human pluripotent stem cells (hPSCs) holds great promise for the understanding and the treatment of human neurological diseases in modern medicine. The hPSCs are considered for their in vitro use as research tools to provide relevant cellular model for human diseases, drug discovery, and toxicity assays and for their in vivo use in regenerative medicine applications. In this review, we highlight recent progress, promises, and challenges of hPSC applications in human neurological disease modeling and therapies.
    Frontiers in Physiology 07/2012; 3:267. DOI:10.3389/fphys.2012.00267 · 3.50 Impact Factor
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